A Melampsora lini β-tubulin gene and flanking sequence has been isolated and characterised. The gene contains 8 introns, is expressed in germinating urediospores and vegetative mycelia and encodes a 448 amino acid protein that is similar to other fungal β-tubulin genes. Evidence for a second β-tubulin gene within the M. lini genome is also presented. The predicted M. lini β-tubulin protein was compared with 34 other fungal β-tubulin proteins in a phylogenetic analysis and, in general, good agreement was observed between the derived β-tubulin based phylogeny and the current taxonomic status of these species. However, a second β-tubulin protein found in five fungal species is atypical when compared to other fungal β-tubulin proteins. Analysis of intron positions within 33 fungal β-tubulin genes showed that intron location was generally characteristic of the fungal class, with euascomycete fungi showing particularly striking conservation of intron position, although intron number was variable and intron sequences showed little conservation.